The development of drug resistant bacteria is a major problem in medicine as more antibiotics are used for a wide variety of illnesses and other conditions. The use of more antibiotics and the number of bacteria showing resistance has prompted longer treatment times. Furthermore, broad, non-specific antibiotics, some of which have detrimental effects on the patient, are now being used more frequently. A related problem with this increased use is that many antibiotics do not penetrate mucus linings easily.
Gram-positive bacteria are surrounded by a cell wall containing polypeptides and polysaccharide. Gram-positive bacteria include but are not limited to the genera Actinomyces, Bacillus, Listeria, Lactococcus, Staphylococcus, Streptococcus, Enterococcus, Mycobacterium, Corynebacterium, and Clostridium. Medically relevant species include Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus, and Enterococcus faecalis. Bacillus species, which are spore-forming, cause anthrax and gastroenteritis. Spore-forming Clostridium species are responsible for botulism, tetanus, gas gangrene and pseudomembranous colitis. Corynebacterium species cause diphtheria, and Listeria species cause meningitis.
Novel antimicrobial therapy approaches include enzyme-based antibiotics (“enzybiotics”) such as bacteriophage lysins. Phages use these lysins to digest the cell wall of their bacterial hosts, releasing viral progeny through hypotonic lysis. A similar outcome results when purified, recombinant lysins are added externally to Gram-positive bacteria. The high lethal activity of lysins against gram-positive pathogens makes them attractive candidates for development as therapeutics (Fischetti, V. A. (2008) Curr Opinion Microbiol 11:393-400; Nelson, D. L. et al (2001) Proc Natl Acad Sci USA 98:4107-4112). Bacteriophage lysins were initially proposed for eradicating the nasopharyngeal carriage of pathogenic streptococci (Loeffler, J. M. et al (2001) Science 294: 2170-2172; Nelson, D. et al (2001) Proc Natl Acad Sci USA 98:4107-4112). Lysins are part of the lytic mechanism used by double stranded DNA (dsDNA) phage to coordinate host lysis with completion of viral assembly (Wang, I. N. et al (2000) Annu Rev Microbiol 54:799-825). Lysins are peptidoglycan hydrolases that break bonds in the bacterial wall, rapidly hydrolyzing covalent bonds essential for peptidoglycan integrity, causing bacterial lysis and concomitant progeny phage release.
Lysin family members exhibit a modular design in which a catalytic domain is fused to a specificity or binding domain (Lopez, R. et al (1997) Microb Drug Resist 3:199-211). Lysins can be cloned from viral prophage sequences within bacterial genomes and used for treatment (Beres, S. B. et al (2007) PLoS ONE 2(8):1-14). When added externally, lysins are able to access the bonds of a Gram-positive cell wall (Fischetti, V. A. (2008) Curr Opinion Microbiol 11:393-400). Bacteriophage lytic enzymes have been established as useful in the assessment and specific treatment of various types of infection in subjects through various routes of administration. For example, U.S. Pat. No. 5,604,109 (Fischetti et al.) relates to the rapid detection of Group A streptococci in clinical specimens, through the enzymatic digestion by a semi-purified Group C streptococcal phage associated lysin enzyme. This enzyme work became the basis of additional research, leading to methods of treating diseases. Fischetti and Loomis patents (U.S. Pat. Nos. 5,985,271, 6,017,528 and 6,056,955) disclose the use of a lysin enzyme produced by group C streptococcal bacteria infected with a C1 bacteriophage. U.S. Pat. No. 6,248,324 (Fischetti and Loomis) discloses a composition for dermatological infections by the use of a lytic enzyme in a carrier suitable for topical application to dermal tissues. U.S. Pat. No. 6,254,866 (Fischetti and Loomis) discloses a method for treatment of bacterial infections of the digestive tract which comprises administering a lytic enzyme specific for the infecting bacteria. The carrier for delivering at least one lytic enzyme to the digestive tract is selected from the group consisting of suppository enemas, syrups, or enteric coated pills. U.S. Pat. No. 6,264,945 (Fischetti and Loomis) discloses a method and composition for the treatment of bacterial infections by the parenteral introduction (intramuscularly, subcutaneously, or intravenously) of at least one lytic enzyme produced by a bacteria infected with a bacteriophage specific for that bacteria and an appropriate carrier for delivering the lytic enzyme into a patient.
Phage associated lytic enzymes have been identified and cloned from various bacteriophages, each shown to be effective in killing specific bacterial strains. U.S. Pat. Nos. 7,402,309, 7,638,600 and published PCT Application WO2008/018854 provides distinct phage-associated lytic enzymes useful as antibacterial agents for treatment or reduction of Bacillus anthraces infections. U.S. Pat. No. 7,569,223 describes lytic enzymes for Streptococcus pneumoniae. Lysin useful for Enterococcus (E. faecalis and E. faecium, including vancomycin resistant strains) are described in U.S. Pat. No. 7,582,291. US 2008/0221035 describes mutant Ply GBS lysins highly effective in killing Group B streptococci. A chimeric lysin denoted ClyS, with activity against Staphylococci bacteria, including Staphylococcus aureus, is detailed in WO 2010/002959.
Based on their rapid, potent, and specific cell wall-degradation and bactericidal properties, lysins have been suggested as antimicrobial therapeutics to combat Gram-positive pathogens by attacking the exposed peptidoglycan cell walls from outside the cell (Fenton, M et al (2010) Bioengineered Bugs 1:9-16; Nelson, D et al (2001) Proc Natl Acad Sci USA 98:4107-4112). Efficacies of various lysins as a single agents have been demonstrated in rodent models of pharyngitis (Nelson, D et al (2001) Proc Natl Acad Sci USA 98:4107-4112), pneumonia (Witzenrath, M et al (2009) Crit Care Med 37:642-649), otitis media (McCullers, J. A. et al (2007) PLOS pathogens 3:0001-0003), abscesses (Pastagia, M et al Antimicrobial agents and chemotherapy 55:738-744) bacteremia (Loeffler, J. M. et al (2003) Infection and Immunity 71:6199-6204), endocarditis (Entenza, J. M. et al (2005) Antimicrobial agents and chemotherapy 49:4789-4792), and meningitis (Grandgirard, D et al (2008) J Infect Dis 197:1519-1522). In addition, lysins are generally specific for their bacterial host species and do not lyse non-target organisms, including human commensal bacteria which may be beneficial to gastrointestinal homeostasis (Blaser, M. (2011) Nature 476:393-394; Willing, B. P. et al (2011) Nature reviews. Microbiology 9:233-243)
Antibiotics in clinical practice include several which commonly affect cell wall peptidoglycan biosynthesis in gram positive bacteria. These include glycopeptides, which as a class inhibit peptidoglycan synthesis by preventing the incorporation of N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) peptide subunits into the peptidoglycan matrix. Available glycopeptides include vancomycin and teicoplanin, with vancomycin a primary drug of choice and clinical application in bacteremia, particularly Staphylococcal infections. Penicillins act by inhibiting the formation of peptidoglycan cross-links. Common penicillins include oxacillin, ampicillin and cloxacillin. Linezolid (Zyvox) is a protein synthesis inhibitor and in a class of antibacterials called oxazolidinones (Ford C W et al (1996) Antimicrob Agents Chemoth 40(6):1508-1513; Swaney S M et al (1998) Antimicrob Agents Chemoth 42(12):3251-3255; U.S. Pat. No. 6,444,813).
Daptomycin (Cubicin), also denoted LY 146032, is a lipopeptide antibacterial agent consisting of a 13-member amino acid peptide linked to a 10-carbon lipophilic tail (Miao V et al (2005) Microbiology 151(Pt5):1507-1523; Steenbergen J N et al (2005) J Antimicrob Chemother 55(3):283-288; and described in U.S. Pat. No. 5,912,226). This structure results in a novel mechanism of action, the disruption of the bacterial membrane through the formation of transmembrane channels, which cause leakage of intracellular ions leading to depolarizing the cellular membrane and inhibition of macromolecular synthesis. Daptomycin's spectrum of activity is limited to Gram-positive organisms, including a number of highly resistant species (methicillin-resistant S. aureus (MRSA), vancomycin intermediate-sensitive S. aureus (VISA), vancomycin-resistant S. aureus (VRSA), vancomycin-resistant Enterococcus (VRE)). In studies it appears to be more rapidly bactericidal than vancomycin. Its approved dosing regimen is 4 mg/kg IV once daily. Dose adjustment is necessary in renal dysfunction. Daptomycin's primary toxicity is reversible dose-related myalgias and weakness. Daptomycin has been approved for the treatment of complicated skin and soft tissue infections caused by gram positive bacteria, Staphylococcus aureus bacteremia and right-sided S. aureus endocarditis. Trials assessing daptomycin's efficacy in treating complicated urinary tract infections and endocarditis/bacteremia are ongoing. Its approved dosing regimen is 4 mg/kg IV once daily. Dose adjustment is necessary in renal dysfunction. Daptomycin's primary toxicity is reversible dose-related myalgias and weakness. Resistance to daptomycin has been encountered both in vitro and in vivo after exposure to daptomycin. The mechanism(s) of resistance are not fully defined but likely relate to alterations of the cellular membrane. Multiple passages of Staphylococci and Enterococci in subinhibitory drug concentrations resulted in MIC increases in a stepwise fashion. Daptomycin binds avidly to pulmonary surfactant and cannot be effectively used in treatment of pneumonia (Baltz R H (2009) Curr Opin Chem Biol 13(2):144-151).
The broad spectrum antibiotics in clinical use for treatment of gram positive infections, particularly including critical care antibiotics such as vancomycin, are limited in use and application by their side effects of gastrointestinal upset and diarrhea and the development of resistance, particularly in connection with continued or long-term use.
It is evident from the deficiencies and problems associated with current traditional antibacterial agents that there still exists a need in the art for additional specific bacterial agents, combinations and therapeutic modalities, particularly without high risks of acquired resistance. Accordingly, there is a commercial need for new antibacterial approaches, especially those that operate via new modalities or provide new combinations to effectively kill pathogenic bacteria.
The citation of references herein shall not be construed as an admission that such is prior art to the present invention.